Internal Transcribed Spacer Ribosomal DNA Research Articles

Non-invasive assessment of parasitic nematode species diversity in wild Soay sheep using molecular markers

Considerable effort has been put into detecting and identifying parasitic nematodes in live ruminants, but to date most studies are limited to a small group of nematodes and/or to experimentally infected sheep. In this study, a PCR-based assay using species-specific primer pairs, located in the second internal transcribed spacer ribosomal DNA, was developed to identify nine different species from six different families of parasitic nematodes in a wild, unmanaged and naturally infected population of sheep. Each primer pair was tested for its specificity and sensitivity and it exclusively amplified the species it was designed for and exhibited a high degree of sensitivity. The method was applied to eggs and cultured larvae to identify the parasitic nematodes present in a pooled faecal sample from several host individuals with unknown parasite burden. To test detection reliability, a faecal sample from an individual with known parasite burden (through post-mortem analysis) was also examined. All species present could be correctly identified by PCR, but detecting very low levels and/or early stages of infection proved to be difficult. The method was also tested for its applicability to high through-put screening of faecal samples.

Linnaeus was right all along: Ulva and Enteromorpha are not distinct genera

Ulva, one of the first Linnaean genera, was later circumscribed to consist of green seaweeds with distromatic blades, and Enteromorpha Link was established for tubular forms. Although several lines of evidence suggest that these generic constructs are artificial, Ulva and Enteromorpha have been maintained as separate genera. Our aims were to determine phylogenetic relationships among taxa currently attributed to Ulva, Enteromorpha, Umbraulva Bae et I.K. Lee and the monotypic genus Chloropelta C.E. Tanner, and to make any nomenclatural changes justified by our findings. Analyses of nuclear ribosomal internal transcribed spacer DNA (ITS nrONA) (29 ingroup taxa including the type species of Ulva and Enteromorphat, the chloroplast-encoded rbcL gene (for a subset of taxa) and a combined data set were carried out. All trees had a strongly supported clade consisting of all Ulva, Enteromorpha and Chloropelta species, but Ulva and Enteromorpha were not monophyletic. The recent removal of Vmbraulva olivascens (PJ.L. Dangeard) Bae et I.K. Lee from Ulvu is supported, although the relationship of the segregate genus Umhraulva to Ulvaria requires further investigation. These results, combined with earlier molecular and culture data, provide strong evidence that Ulva, Enteromorpha and Chloropelta are not distinct evolutionary entities and should not be recognized as separate genera. A comparison of traits for surveyed species revealed few synapomorphies. Because Ulva is the oldest name, Enteromorpha and Chloropclta are here reduced to synonymy with Ulva, and new combinations are made where necessary.

Kinetoplastid Internet Symposium: A KISs for 2002.

Wanderley de Souza (Universidade Federal do Rio de Janeiro, Brazil) offered to head up the scientific agenda with a comprehensive description of how breakthroughs in the study of trypanosome cell biology could be translated into new drug targets and into affirmative action in combating disease. Kwang-Poo Chang (Chicago Medical School, IL, USA) and Brad McGwire (University of Illinois, IL, USA) focused on pathoantigenic determinants and their role in determining the virulence and pathogenesis of leishmaniasis, particularly on the complex interactions between multiple determinants, which can lead to various outcomes. The online discussion forum was then used to explore the importance of these antigenic determinants in the fabrication of immunoprophylactics, especially with regard to live attenuated Leishmania vaccines.Serap Aksoy (Yale University School of Medicine, CT, USA), Zhengrong Hao (Yale University School of Medicine) and Patricia Strickler (Yale University School of Medicine) authored an intriguing discussion on the importance of molecular studies directed at dissecting tsetse–trypanosome interactions. The response role of insect defensins and the possible use of paratransgenic tsetse with transformed Wolbachia (rendering tsetse refractory to trypanosome infection) were discussed in the article and online.Joao Carlos Dias (Oswaldo Cruz Foundation, Belo Horizonte, Brazil) and Christopher Schofield (London School of Hygiene and Tropical Medicine, UK) presented a fascinating report highlighting the current status of Chagas disease control throughout Latin America. The report emphasized the need to capitalize on recent gains made by regional programs, such as the Southern Cone Initiative, to prevent the disease re-emerging in areas where transmission has effectively been eliminated. Moreover, such programs should be extended into regions where the disease is still endemic. In particular, the report highlighted research areas that are most likely to produce significant steps forward in combating Chagas disease including triatomine sampling, migratory behavior and epidemiological analysis, improved serological tests, new drug designs and analysis of regional program structures.Finally, a strong poster session covering advances from the fields of diagnostics and chemotherapy was presented online. One significant presentation from Wendy Gibson et al. (University of Bristol, UK) described markers that identified Trypanosoma brucei rhodesiense from other members of the Trypanosoma brucei complex. Other presentations covered the phylogenetic identity of Leishmania mexicana (Amalia Monroy-Ostria and Omar Hernandez-Montes, Col. Casco de Santo Tomas, Mexico) and the T. brucei genome initiative using single nucleotide polymorphisms (SNPs) suitable for population analysis in African trypanosomes (Emmanuelle Bart-Delabesse et al., University of Cambridge, UK). The use of internal transcribed spacer ribosomal DNA (ITS-rDNA) for diagnosing Trypanosoma lewisi in laboratory animals was also described by Marc Desquesnes and colleagues (Centre International de Recherche-Developpement sur l'Elevage en zone Subhumide, Burkina Faso). Posters on veterinary chemotherapeutics by Alain Bourdichon et al. (Atarost, Hamburg, Germany) discussed the efficacy of diminazene against Trypanosoma evansi in combination with antipyrine or Trypan (procaine) and using liposomes as a delivery vehicle.Until the launch of Kinetoplastid Biology and Disease, articles from the conference will remain online at http://www.trypanosome.com We invite volunteers interested in co-ordinating further Internet meetings or developing integrated Internet resources for kinetoplastid diseases to contact us.

Development of a PCR‐based diagnostic test for Spongospora subterranea f.sp. nasturtii, the causal agent of crook root of watercress (Rorippa nasturtium‐aquaticum)

The polymerase chain reaction (PCR) technique was utilized to obtain internal transcribed spacer ribosomal DNA (ITS rDNA) and small‐subunit (18S) rDNA sequences from UK isolates of Spongospora subterranea f.sp. nasturtii, a plasmodiophorid pathogen of watercress (Rorippa nasturtium‐aquaticum). ITS sequence data obtained from S. subterranea isolated from a range of UK sites were found to be identical. PCR primers were designed using these sequences and were shown to be capable of specific amplification of S. subterranea f.sp. nasturtii DNA from plant tissue and from water samples containing zoospores of the pathogen. As little as 5 ng total genomic DNA from infected plant material, or 1000 zoospores, was required for consistently successful amplification of DNA. A filtration‐based method for obtaining pathogen DNA for PCR from watercress‐bed water was developed.

Phylogenetic analysis of Polymyxa species based on nuclear 5.8S and internal transcribed spacers ribosomal DNA sequences

A region of the nuclear ribosomal DNA containing the internal transcribed spacer 1 (ITS1), the 5.8S DNA and the internal transcribed spacer 2 (ITS2) was sequenced in 12 Polymyxa graminis and P. betae isolates, with particular emphasis on P. graminis from peanut clump-infested areas of the Indian subcontinent and West-Africa. Four different sequences were obtained from the seven isolates on sorghum or pearl millet, which differed from four sequences previously published for Polymyxa species and obtained for P. graminis isolates on barley, oat and wheat originating from temperate areas in Europe and America (two distinct sequences), for isolates on rice from Colombia and for P. betae isolates on sugar beet from several origins. The sequence variations concerned mainly the composition and length of ITS1 and ITS2 regions. Phylogenetic trees built with the distinct sequences currently known for Polymyxa spp. using parsimony, maximum likeihood and neighbour-joining methods separated P. betae from P. graminis, Within P. graminis, the hierarchy of the clustering partially matched the host range and geographical origin of the isolates. These results confirm the great diversity within P. graminis that has already been observed in host range and temperature requirements studies, and provide new arguments for considering several taxa within the species. On the basis of the ecological requirements and rDNA sequences of distinct P. graminis isolates, five special forms are proposed: P. graminis f. sp. temperata, P. graminis f. sp. tepida, P. graminis f. sp. subtropicalis, P. graminis f. sp. tropicalis and P. graminis f. sp. colombiana.

Phylogeography of the ectomycorrhizal Pisolithus species as inferred from nuclear ribosomal DNA ITS sequences

Summary The fungal genus Pisolithus is cosmopolitan in warm temperate regions and forms ectomycorrhizal associations with a wide range of woody plants. To delimit phylogenetic Pisolithus species and identify their geographical distribution, 102 collections were made worldwide and their rDNA internal transcribed spacer (ITS) was sequenced. Phylogenetic analyses of these sequences, together with 46 additional GenBank accessions, identified 11 species. A strong phylogeographical pattern was observed related to the native geographical origin of the host plants. In the Holarctic, P. tinctorius was widely distributed, associated with Pinus and Quercus. It has been co‐introduced with pines to other biogeographical regions. Several Pisolithus lineages, including P. aurantioscabrosus, occurred in restricted biogeographical regions associated with endemic plants, such as Afzelia in eastern Africa. Pisolithus albus, P. marmoratus and P. microcarpus were associated with Australasian hosts (Eucalyptus, Acacia) and were distributed with their hosts worldwide. By contrast, two additional unnamed species were restricted to Australia. The present study shows that evolutionary lineages within Pisolithus are related to the biogeographical origin of the hosts. In addition, regional floras and endemic plants could act as hosts of endemic species of Pisolithus.

Sequence analysis of the ribosomal internal transcribed spacer DNA of the crayfish parasite Psorospermium haeckeli.

Two morphotypes of the crayfish parasite Psorospermium haeckeli were isolated from 2 crayfish species of different geographical origin. The oval-shaped sporocysts were obtained from the epidermal and connective tissue beneath the carapace of the noble crayfish Astacus astacus from Sweden and Finland. Elongated spores were isolated from the abdominal muscle tissue of the red swamp crayfish Procambarus clarkii from USA. To compare genetic divergence of 2 morphotypes of the parasite, the ribosomal internal transcribed spacer (ITS) DNA (ITS 1 and ITS 2) and the 5.8S rRNA gene were cloned and sequenced. The analysed region is variable in length, with the ribosomal ITS sequence of the European morphotype longer than the North American one. Sequence diversity is found mainly in ITS 1 and ITS 2 regions, and there is 66% and 58% similarity between the 2 morphotypes, respectively. Thus, analysis of the ribosomal ITS DNA suggests that P. haeckeli forms obtained from Europe and North America are genetically diverse, which supports the previously reported morphological characteristics.

THE PHYLOGENY OF CAULERPA BASED ON RDNA INTERNAL TRANSCRIBED SPACER SEQUENCES

Phylogenetic hypotheses for the pantropical marine green algal genus, Caulerpa, were inferred based on analyses of nuclear‐encoded rDNA internal transcribed spacer (ITS) sequences. Results of these analyses were used to assess the correspondence between rDNA phylogeny and traditional sectional taxonomy, to identify synapomorphic morphological characters (including assimilator morphology and chloroplast ultrastructure), and to examine marine biogeographic hypotheses for the genus. Ribosomal DNA ITS sequences were aligned for thirty‐three species and intraspecific taxa of Caulerpa. Results indicate limited correspondence between phylogeny and sectional taxonomy for the genus, (e.g., the sections Filicoideae and Sedoideae were not monophyletic). In contrast, chloroplast morphology could be mapped to the tree topology with limited homoplasy. Pantropical isolates of the filicoidean species, Caulerpa sertularioides and Caulerpa mexicana each formed monophyletic groups. Caulerpa reyesii was included as a derived taxon within the Caulerpa taxifolia clade, suggesting that these species were conspecific and affirmed the lack of correspondence between phylogeny and assimilator morphology. Isolates and various intraspecific taxa of Caulerpa racemosa did not form a monophyletic group. Instead, these taxa formed a heterogeneous assemblage with other sedoidean and filicoidean taxa. Within the C. sertularioides clade, Caribbean and Atlantic isolates formed a basal paraphyletic group, whereas eastern and western Pacific isolates formed a more derived monophyletic group. Therefore, these results are not consistent with an Indo‐West Pacific origin of this species.

Internal transcribed spacer ribosomal DNA sequence of five species of Ganoderma from Australia

Phylogeny inferred from ITS DNA sequence resolved isolates of Australian Ganoderma into five terminal clades. The phylogenetic analysis was considered superior to sequence variation statistics for delineating species as intra and inter-specific variation were dissimilar across the taxa studied. The analysis also revealed that a number of isolates had been misnamed. Phylogeny inferred from the sequence data supported the retention of Elfvingia and Ganoderma as subgenera of Ganoderma, and the placement of the G. chalceum complex within subgenus Ganoderma. These findings were, however, poorly supported by decay analysis. G. adspersum, a European species considered to be a synonym of G. australe (a species which is in need of neotypification) by some authors, was not synonymous with G. australe from Australia. G. incrassatum from Northern Australia formed a clade with isolates named G. gibbosum from China and an isolate named G. australe from Taiwan. G. weberianum from Australia, originally identified by R. L. Steyaert, formed a terminal clade that was closely related to a second terminal clade comprised of two G. weberianum isolates and an isolate of G. microsporum from Asia. It was concluded that the clades represented two allopatric populations of G. weberianum and that G. microsporum was a synonym of G. weberianum. Isolates determined as G. chalceum by R. L. Steyaert and referred to here as G. cupreum formed a distinct terminal clade most closely related to material identified as G. sinense. A species mistaken for G. lucidum from Australia and Indonesia, a species from temperate and South East Asia containing commercially cultivated strains, and an Argentinean species showed evidence of allopatric speciation. Nomenclatural problems and the implications of our study on the pharmaceutical and commercial exploitation of Ganoderma in Australia are discussed.

Sequence differences in the internal transcribed spacer ribosomal DNA among four species of hookworm (Ancylostomatoidea: Ancylostoma)

The two ribosomal DNA internal transcribed spacers (1 and 2) of the hookworms Ancylostoma caninum, A. tubaeforme, A. ceylanicum and A. duodenale were sequenced. The sequence lengths were similar among the four species, except that A. ceylanicum had slightly longer (by 5–7 bp) internal transcribed spacer 1 and 2 sequences. The predicted secondary structure of the internal transcribed spacer 2 precursor rRNA was similar for all species, despite interspecific differences in primary sequence ranging from 0.9% to 13.2%. Interspecific differences in internal transcribed spacer 1 sequence ranged from 0.9% to 7.5%. A cladistic analysis of the sequence data, using the human hookworm Necator americanus as the outgroup, provided little resolution of the phylogenetic relationships, except that A. ceylanicum occurred on a branch external to the other three species. Nonetheless, internal transcribed spacers 1 and 2 may provide useful phylogenetic information at higher taxonomic levels within the superfamily Ancylostomatoidea.

Phylogeny of Bursaphelenchus species derived from analysis of ribosomal internal transcribed spacer DNA sequences

Abstract Bursaphelenchus species can cause serious economic damage to pine forestry, and are widely distributed across the globe. The genetic structure of the B. mucronatus-B. xylophilus species group was investigated using sequences of the internal transcribed spacers of the ribosomal RNA gene repeat as a marker. Analysis of the ITS from 11 Bursaphelenchus isolates, using Aphelenchoides rhytium as an outgroup, supports the perception that the two species are distinct genetic entities. Significant divergence was however found within each species. ITS sequence analysis does not support a separation of European and Japanese B. mucronatus into distinct species-level taxa. Phylogenie des especes de Bursaphelenchus issue d'une analyse des sequences du DNA ribosomique de l'espaceur interne transcrit - Les especes de Bursaphelenchus peuvent provoquer de serieux degats aux cultures de pins et sont largement reparties dans le monde. La structure genetique des especes du groupe B.mucronatus-B. xylophilus a ete etudiee en utilisant comme marqueur des sequences repetees de l'espaceur interne transcrit du gene de l'ARN ribosomique. L'analyse des ITS de 11 isolats de Bursaphelenchus, en utilisant Aphelenchoides rhytium comme un extra-groupe, conforte l'idee que les deux especes sont des entites genetiques distinctes. Une divergence significative a cependant ete trouvee a l'interieur de chaque especes. L'analyse des sequences des ITS n'accredite pas la separation des B. mucronatus europeen et japonais en deux especes distinctes.

Molecular phylogeny of Mesometridae (Trematoda, Digenea) with its relation to morphological changes in parasites.

Complete ITS (Internal Transcribed Spacer) ribosomal DNA sequences were obtained for the six species know at present time within the Mesometridae Poche, 1926. The adult stages are intestinal parasites of herbivorous sparid teleosts. Aligned sequences were analysed with Maximum Parsimony, Maximum Likelihood and Neighbor-Joining phylogenetic methods to infer evolutionary relationships among mesometrid species. The ITS-based phylogeny obtained showed the two Wardula species as a sister group to other Mesometridae, and as compared to morphological data, suggest some general tendencies in the morphological evolution of this group. It consists mainly in changes from elongated to subcircular forms, regression of the pharynx, and the development of a strong accessory attachment organ.

Common secondary structures for the second internal transcribed spacer pre-rRNA of two subfamilies of trichostrongylid nematodes

Sequences of the second internal transcribed spacer ribosomal DNA for the parasitic trichostrongylid nematodes Trichostrongylus probolurus, Trichostrongylus rugatus and Camelostrongylus mentulatus were compared with previously published sequences for five other species within the genus Trichostrongylus. The secondary structures of the second internal transcribed spacer pre-rRNA for these nematodes were predicted using an energy minimisation method. The results indicate that a common secondary structure of the second internal transcribed spacer of these nematodes is maintained despite distinct differences in primary sequence between species. Sequence differences among Trichostrongylus species ranged from 1.3 to 7.6%, but each species differed by 22–26% in sequence when compared with C. mentulatus which belongs to a different subfamily.

Use of the ITS rDNA for elucidation of some life-cycles of Mesometridae (Trematoda, Digenea)

Identification of larval stages is crucial for elucidating the life-cycles of various Digenea. However, in many digenean species, the larvae lack distinctive morphological features and it is impossible to establish the affiliation between the larval and adult stages by using morphological criteria. Molecular methods, based on DNA sequencing or PCR–restriction fragment length polymorphism analysis, can offer a new tool for larval-stage identification. In this study, the sequences of internal transcribed spacer of the ribosomal DNA were used to identify the cercariae of three out of five species of the family Mesometridae ( Centroderma spinosissima, Elstia stossichianum and Wardula capitellata). The three species differ from one another by number of repeats in the region of internal transcribed spacer 1. The phylogeny of Mesometridae was inferred from their internal transcribed spacer ribosomal DNA sequences. The PCR-linked restriction fragment length polymorphism approach was developed for future life-cycle and ecological studies of this family.

Variation in the ITS and IGS regions of ribosomal DNA among the biological species of European Armillaria

Variation within the internal transcribed spacer (ITS) and the intergenic spacer (IGS) of the ribosomal RNA gene of isolates representing seven European species of Armillaria was examined by PCR, coupled with RFLP analysis and partial sequencing of the ITS region.

PCR-SSCP Analysis of the Ribosomal DNA ITS Regions of the Willow Rust Fungi in Japan.

Single-strand DNA conformation polymorphism (SSCP) analysis using internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) amplified by polymerase chain reaction (PCR) was performed to detect DNA polymorphism of nine species of Melampsora rust fungi on willows. The rDNA ITS 1 region (approximately 300-320bp in size), was amplified from 49 collections. The SSCP patterns in the amplified ITS 1 region of five species, M. capraearum, M. epiphylla, M. larici-urbaniana, M. microsora and M. yezoensis were species-specific. On the other hand, no distinction was found in the SSCP patterns between M. chelidonii-pierotii and M. coleosporioides, and between M. epitea and M. humilis. In improved PCR-SSCP analysis using restriction fragments of the amplified ITS 1 and ITS 2 regions, no difference was detected between M. chelidonii-pierotii and M. coleosporioides, and between M. epitea and M. humilis. These results suggest the respective two species have a close genetic and taxonomical relationship. We revealed the PCR-SSCP analysis may be beneficial as a tool for distinguishing species of Melampsora because the analysis is simple and sensitive.

An examination of structural features and relationships in Balansia subgenus Dothichloë

Balansia subgenus Dothichloë is defined to include only species of graminicolous Clavicipitaceae that are endophytic, form stromata on leaves or culms, and produce nonstipitate black ascomata. Species included in subgenus Dothichloë are Balansia aristidae, B. epichloë, B. henningsiana, B. nigricans, B. strangulans, and Balansiopsis gaduae. The genus Balansiopsis is invalidated since its type B. gaduae is herein classified in subgenus Dothichloë. Balansia subnodosa is considered to be a synonym of B. gaduae based on morphological and anatomical similarities of the ascomata. Phylogenetic relationships in Dothichloë are evaluated through examination of ribosomal DNA ITS (internal transcribed spacer) sequence data. It is concluded that at least two subclades are evident in the subgenus, including the B. gaduae/B. nigricans subclade and the B. aristidae/B. henningsiana/B. strangulans subclade. Sequence divergence of B. epichloë makes it difficult to confidently assign it to either subclade. A dichotomous key to Dothichloë is included along with descriptions of B. aristidae, B. epichloë, B. gaduae, and B. henningsiana.

An Examination of Structural Features and Relationships in Balansia Subgenus Dothichloe

Balansia subgenus Dothichloë is defined to include only species of graminicolous Clavicipitaceae that are endophytic, form stromata on leaves or culms, and produce nonstipitate black ascomata. Species included in subgenus Dothichloë are Balansia aristidae, B. epichloë, B. henningsiana, B. nigricans, B. strangulans, and Balansiopsis gaduae. The genus Balansiopsis is invalidated since its type B. gaduae is herein classified in subgenus Dothichloë. Balansia subnodosa is considered to be a synonym of B. gaduae based on morphological and anatomical similarities of the ascomata. Phylogenetic relationships in Dothichloë are evaluated through examination of ribosomal DNA ITS (internal transcribed spacer) sequence data. It is concluded that at least two subclades are evident in the subgenus, including the B. gaduae/B. nigricans subclade and the B. aristidae/B. henningsiana/B. strangulans subclade. Sequence divergence of B. epichloë makes it difficult to confidently assign it to either subclade. A dichotomous key to Dothichloë is included along with descriptions of B. aristidae, B. epichloë, B. gaduae, and B. henningsiana.

Molecular phylogenetics ofThlaspis.l. (Brassicaceae) based on chloroplast DNA restriction site variation and sequences of the internal transcribed spacers of nuclear ribosomal DNA

Systematics of the genus Thlaspi s.l. is difficult and controversial. Previous hypotheses have been based on morphological and anatomical data. We have analyzed sequence variation of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (nrDNA) among 13 Thlaspi s.l. taxa, representing all sections of the genus. Phylogenetic relationships among ITS sequences of the Thlaspi s.l. taxa studied are in general concordance with a previously published chloroplast DNA based phylogeny of this group. Most-parsimonious trees from ITS and chloroplast DNA data support three groups that are congruent with lineages (Thlaspi s.str., Noccaea–Raparia, Microthlaspi) previously described by Meyer on the basis of seed anatomy. The ITS data grouped Microthlaspi granatense outside the Microthlaspi clade and, therefore, Microthlaspi appeared paraphyletic on the ITS tree, in contrast with the chloroplast DNA phylogeny. We speculate that concerted evolutionary forces have acted among different nrDNA arrays (brought together in M. granatense by hybridization with a related taxon), resulting in the fixation of the alien species nrDNA type in M. granatense, which, however, maintains a Microthlaspi chloroplast genome type. Both molecular data sets detected intraspecific variation among Microthlaspi perfoliatum accessions of different geographic origin and different ploidy levels. Our molecular evidence would suggest the hybrid origin of polyploid M. perfoliatum from diploid M. perfoliatum and M. natolicum. Key words: chloroplast DNA, restriction site variation, sequence analysis of nuclear ribosomal DNA ITS regions, Thlaspi s.l. (Brassicaceae), molecular phylogeny, congruence.

The genus Tephrocybe Donk in Britain

Tricholoma dulciolens is an ectomycorrhizal basidiomycete that produces “matsutake”-like mushrooms in association with Picea abies in Fennoscandia. The phylogenetic position of T. dulciolens relative to matsutake species is, however, unknown. In the present study, we demonstrated that T. dulciolens is phylogenetically independent from T. matsutake and its allied species. In addition, herbarium specimens identified as T. caligatum from Abies alba forests in Spain were phylogenetically related to but distinct from T. dulciolens. Based on ribosomal internal transcribed spacer DNA sequence data, T. dulciolens appears to exist in North America, as well. This is the first phylogeny of T. dulciolens in Fennoscandia and its related species in southern Europe.